Objectives: This study sought to investigate the diffusion of PCN through the pulmonary membrane and identify any potential barriers to diffusion. This was followed by studies of mice and human blood following exposure to PCN. Materials and Methods: Permeability and metabolism were assessed in silica. C57BL/6J male mice were inoculated by intranasal route with 50μg PCN, and plasma levels were detected by validated HPLC method. Open field and forced swimming test were performed to assess the effect of PCN on mouse behaviour. PCN in CF patients chronically infected withP. aeruginosawas determined by HPLC. Results: This study found that PCN is readily diffusible in silica in a concentration-pH-dependent manner. PCN was not readily metabolised by CYP enzymes in silica and demonstrated to be NADP+ dependent. Significant levels of PCN were detected in mice plasma samples following short-term exposure, and in the sputum of some samples from subjects chronically exposed to PCN producing P. aeruginosa. Our data demonstrated that PCNcan diffuse from the lungs into systemic circulation following both chronic and acute exposure. No significant barriers to diffusion exist. Conclusion: This study provides novel evidence for the role of pyocyanin in systemic damage in cystic fibrosis.